Sample observation device and sample observation method

ABSTRACT

A sample observation device (1) includes: an emission optical system (3) for emitting planar light (L2) onto a sample (S); a scanning unit (4) for scanning the sample (S) with respect to an emission face (R) of the planar light (L2); an imaging optical system (5) having an observation axis (P2) inclined with respect to the emission face (R) and for forming an image from observation light (L3) generated in the sample (S) in accordance with the emission of the planar light (L2); an image acquiring unit (6) for acquiring a plurality of partial image data corresponding to a part of an optical image according to the observation light (L3) formed as an image by the imaging optical system (5); and an image generating unit (8) for generating observation image data of the sample S based on the plurality of partial image data generated by the image acquiring unit (6).

TECHNICAL FIELD

The present disclosure relates to a sample observation device and asample observation method.

BACKGROUND ART

As a method for observing the inside of a sample having athree-dimensional stereoscopic structure such as a cell, selective planeillumination microscopy (SPIM) is known. For example, a tomographicimage observation device described in Patent Document 1 discloses thebasic principle of SPIM of emitting planar light to a sample, forming animage of fluorescent light or scattered light generated inside thesample on an imaging surface, and acquiring image data observed frominside the sample.

As another sample observation device using planar light, for example,there is an SPIM microscope described in Patent Document 2. In thisconventional SPIM endoscope, planar light is emitted while maintaining aconstant inclination angle with respect to an arrangement face of asample, and observation light from the sample is captured by anobservation optical system having an observation axis orthogonal to anemission face of the planar light.

CITATION LIST Patent Literature

Patent Literature 1: Japanese Unexamined Patent Publication No.S62-180241

Patent Literature 2: Japanese Unexamined Patent Publication No.2014-202967

SUMMARY OF INVENTION Technical Problem

In the sample observation device described in Patent Document 2described above, by emitting planar light onto the whole surface of afocused face of an observation optical system, an image of a fault planein an observation axial direction can be acquired by performing imagingonce. Accordingly, in order to acquire three-dimensional information ofa sample, it is necessary to scan the sample in the observation axialdirection and acquire images of a plurality of fault planes in theobservation axial direction. In such a conventional sample observationdevice, it is necessary to repeat selection of a fault plane from whichan image is acquired (scanning a sample and stopping) and imageacquisition until the images of all the fault planes are acquired. Inaddition, in a case in which an area occupied by an observation targetis larger than an imaging area, in addition to the operation ofacquiring a cross-sectional image in the observation axial direction,operations of moving a stage in a direction different from theobservation axial direction and selecting an imaging field of view andthe like are necessary. For this reason, there is a problem in that ittakes time until observation image data is acquired.

An object of an embodiment is to provide a sample observation device anda sample observation method.

Solution to Problem

According to one aspect of an embodiment, there is provided a sampleobservation device including: an emission optical system for emittingplanar light onto a sample; a scanning unit for scanning the sample withrespect to an emission face of the planar light; an imaging opticalsystem having an observation axis inclined with respect to the emissionface and for forming an image from observation light generated in thesample in accordance with the emission of the planar light; an imageacquiring unit for acquiring a plurality of partial image datacorresponding to a part of an optical image according to the observationlight formed as an image by the imaging optical system; and an imagegenerating unit for generating observation image data of the samplebased on the plurality of partial image data generated by the imageacquiring unit.

In this sample observation device, a sample is scanned by the emissionface of the planar light, and the observation axis of the imagingoptical system is inclined with respect to the emission face of theplanar light. For this reason, the image acquiring unit can sequentiallyacquire partial image data of fault planes in the direction of theoptical axis of the planar light, and the image generating unit cangenerate observation image data of the sample based on the plurality ofpartial image data. In this sample observation device, an operation ofselecting a field of view is not necessary, and a scanning operation fora sample and image acquisition can be simultaneously performed, wherebythe throughput until the acquisition of the observation image data isimproved.

In addition, the sample may be held by a sample container having aninput face of the planar light, and an optical axis of the planar lightaccording to the emission optical system may be disposed to beorthogonal to the input face of the sample container. In such a case, aplurality of samples can be scanned together using the sample container.In addition, by configuring the optical axis of the planar light to beorthogonal to the input face of the sample container, positioncorrection for the partial image data acquired by the image acquiringunit and the like are not necessary, and the process of generatingobservation image data can be easily performed.

In addition, the scanning unit may scan the sample in a directionorthogonal to the optical axis of the planar light according to theemission optical system. In such a case, image processing such asposition correction for the partial image data acquired by the imageacquiring unit and the like is not necessary, and the process ofgenerating observation image data can be easily performed.

In addition, an inclination angle of the observation axis of the imagingoptical system with respect to the emission face of the planar light maybe in the range of 10° to 80°. In this range, the resolution of anobserved image can be sufficiently secured.

Furthermore, an inclination angle of the observation axis of the imagingoptical system with respect to the emission face of the planar light maybe in the range of 20° to 70°. In this range, the resolution of anobserved image can be more sufficiently secured. In addition, a changein the field of view with respect to the amount of change in the angleof the observation axis can be suppressed, and the stability of thefield of view can be secured.

In addition, an inclination angle of the observation axis of the imagingoptical system with respect to the emission face of the planar light maybe in the range of 30° to 65°. In this range, the resolution of anobserved image and the stability of the field of view can be moreappropriately secured.

In addition, the image acquiring unit may be configured to include atwo-dimensional imaging device and for extracting image datacorresponding to a part of the optical image of the observation lightfrom data output from the two-dimensional imaging device as the partialimage data. According to such a configuration, the partial image datacan be acquired with high accuracy.

In addition, the image acquiring unit may include a line sensor forcapturing a part of the optical image according to the observation lightand outputting the partial image data. According to such aconfiguration, the partial image data can be acquired with highaccuracy.

In addition, the image acquiring unit may include a slit transmitting apart of an optical image according to the observation light and anoptical detector for detecting an optical image transmitted through theslit and is configured to generate the partial image data based on dataoutput from the optical detector. According to such a configuration, thepartial image data can be acquired with high accuracy.

In addition, the image generating unit may be configured to generateobservation image data of the sample on a face orthogonal to the opticalaxis of the planar light based on the plurality of partial image data.In such a case, a cross-sectional image of the sample in which theinfluence of background is suppressed can be acquired as an observedimage.

In addition, the sample observation device may further include ananalysis unit for analyzing the observation image data and generating ananalysis result. Since the observation image data generated by the imagegenerating unit is analyzed by the analysis unit, the throughput of theanalysis can be improved as well.

In addition, according to one aspect of an embodiment, there is provideda sample observation method including: an emission step of emittingplanar light onto a sample; a scanning step of scanning the sample withrespect to an emission face of the planar light; an imaging step offorming an image from observation light generated in the sample inaccordance with the emission of the planar light using an imagingoptical system having an observation axis inclined with respect to theemission face; an image acquiring step of acquiring a plurality ofpartial image data corresponding to a part of an optical image accordingto the observation light formed as an image by the imaging opticalsystem; and an image generating step of generating observation imagedata of the sample based on the plurality of partial image data.

In this sample observation method, a sample is scanned by the emissionface of the planar light, and the imaging optical system of which theobservation axis is inclined with respect to the emission face of theplanar light is used. For this reason, in the image acquiring step,partial image data of fault planes in the direction of the optical axisof the planar light can be sequentially acquired, and in the imagegenerating step, observation image data of the sample can be generatedbased on a plurality of partial image data. In this sample observationmethod, an operation of selecting a field of view is not necessary, anda scanning operation for a sample and image acquisition can besimultaneously performed, whereby the throughput until the acquisitionof the observation image data is improved.

Advantageous Effects of Invention

According to a sample observation device and a sample observationmethod, throughput until acquisition of observation image data isimproved.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic configuration diagram illustrating a sampleobservation device according to one embodiment.

FIG. 2 is a main-part enlarged diagram illustrating the vicinity of asample.

FIG. 3 is a diagram illustrating one example of an image acquiring unit.

FIG. 4 is a flowchart illustrating one example of a sample observationmethod using a sample observation device.

FIG. 5 is a diagram illustrating one example of generation ofobservation image data using an image generating unit.

FIG. 6 is a diagram illustrating a view of image acquisition in acomparative example.

FIG. 7 is a diagram illustrating a view of image acquisition accordingto an example.

FIG. 8 is a diagram illustrating an example of calculation of a field ofview in a sample observation device.

FIG. 9 is a diagram illustrating a relation between an inclination angleof an observation axis and resolution.

FIG. 10 is a diagram illustrating a relation between an inclinationangle of an observation axis and stability of a field of view.

FIG. 11 is a diagram illustrating a relation between an inclinationangle of an observation axis and transmittance of observation lighttransmitted from a sample.

FIG. 12 is a diagram illustrating a modified example of an imagingoptical system.

DESCRIPTION OF EMBODIMENT

Hereinafter, a sample observation device and a sample observation methodaccording to a preferred embodiment will be described in detail withreference to the drawings.

[Configuration of Sample Observation Device]

FIG. 1 is a schematic configuration diagram illustrating a sampleobservation device according to one embodiment. This sample observationdevice 1 is a device that emits planar light L2 onto a sample S andacquires observation image data of the inside of the sample S by formingan image using at least one of fluorescent light, scattered light, anddiffuse reflected light generated inside the sample S on an imagingsurface. As a sample observation device 1 of such a type, there is aslide scanner that acquires and displays an image of a sample Smaintained in a slide glass, a plate reader that acquires image data ofa sample S maintained on a microplate and analyzes the image data, orthe like. The sample observation device 1, as illustrated in FIG. 1, isconfigured to include a light source 2, an emission optical system 3, ascanning unit 4, an imaging optical system 5, an image acquiring unit 6,and a computer 7.

As a sample S that is an observation target, for example, there is acell, a tissue, or an organ of a human or an animal, an animal or aplant, a cell or a tissue of a plant, or the like. In addition, thesample S may be contained in a solution, a gel, or a substance of whicha refractive index is different from that of the sample S.

The light source 2 is a light source that outputs light L1 to be emittedto the sample S. As the light source 2, for example, there is a laserlight source such as a laser diode or a solid laser light source. Inaddition, the light source 2 may be a light emitting diode, a superluminescent diode, or a lamp-system light source. The light L1 outputfrom the light source 2 is guided to the emission optical system 3.

The emission optical system 3 is an optical system that shapes the lightL1 output from the light source 2 into planar light L2 and emits theshaped planar light L2 onto the sample S along an optical axis P1. Inthe following description, the optical axis P1 of the emission opticalsystem 3 may be referred to as an optical axis of the planar light L2.The emission optical system 3, for example, is configured to include alight shaping device such as a cylindrical lens, an axicon lens, or aspatial light modulator and is optically coupled with the light source2. The emission optical system 3 may be configured to include anobjective lens. The planar light L2 formed by the emission opticalsystem 3 is emitted to the sample S. In the sample S to which the planarlight L2 is emitted, observation light L3 is generated on an emissionface R of the planar light L2. The observation light L3, for example, isat least one of fluorescent light excited by the planar light L2,scattered light of the planar light L2, and diffuse reflected light ofthe planar light L2.

In a case in which observation is performed in a thickness direction ofthe sample S, it is preferable that the planar light L2 be thin planarlight of which a thickness is 2 mm or less in consideration of theresolution. In addition, in a case in which the thickness of the sampleS is very small, in other words, in a case in which a sample S having athickness that is equal to or less than Z-direction resolution isobserved, there is no influence of the thickness of the planar light L2on the resolution. Accordingly, planar light L2 of which a thicknessexceeds 2 mm may be used.

The scanning unit 4 is a mechanism that scans the sample S with theemission face R of the planar light L2. In this embodiment, the scanningunit 4 is configured of a moving stage 12 that moves a sample container11 holding the sample S. The sample container 11, for example, is amicro plate, a slide glass, a petri dish, or the like. In thisembodiment, the micro plate will be illustrated as an example. Thesample container 11, as illustrated in FIG. 2, includes a plate-shapedmain body part 14 in which a plurality of wells 13 in which the sample Sis disposed are arranged in one straight line pattern (or a matrixpattern) and a plate-shaped transparent member 15 disposed to close oneend side of the wells 13 on one face side of the main body part 14.

In arranging a sample S inside a well 13, the inside of the well 13 maybe filled with a medium such as water. The transparent member 15includes an input face 15 a of the planar light L2 for the samplearranged inside the well 13. The material of the transparent member 15is not particularly limited as long as it is a member havingtransparency for the planar light L2 and, for example, is glass,crystal, or a synthetic resin. The sample container 11 is arranged withrespect to the moving stage 12 such that the input face 15 a isorthogonal to the optical axis P1 of the planar light L2. In addition,the other end side of the well 13 is open to the outside. The samplecontainer 11 may be fixed to the moving stage 12.

The moving stage 12, as illustrated in FIG. 1, scans the samplecontainer 11 in a direction set in advance in accordance with a controlsignal supplied from the computer 7. In this embodiment, the movingstage 12 scans the sample container 11 in one direction within a planeorthogonal to the optical axis P1 of the planar light L2. In thefollowing description, the direction of the optical axis P1 of theplanar light L2 will be referred to as a Z axis, a scanning direction ofthe sample container 11 according to the moving stage 12 will bereferred to as a Y axis, and a direction orthogonal to the Y axis withinthe plane orthogonal to the optical axis P1 of the planar light L2 willbe referred to as an X axis. The emission face R of the planar light L2for the sample S is a face within an XY plane.

The imaging optical system 5 is an optical system that images theobservation light L3 generated in the sample S in accordance withemission of the planar light L2. The imaging optical system 5, asillustrated in FIG. 2, for example, is configured to include anobjective lens 16, an imaging lens, and the like. An optical axis of theimaging optical system 5 becomes an observation axis P2 of theobservation light L3. The observation axis P2 of this imaging opticalsystem 5 is inclined with respect to the emission face R of the planarlight L2 in the sample S at an inclination angle θ. The inclinationangle θ coincides with an angle formed by the optical axis P1 of theplanar light L2 toward the sample S and the observation axis P2. Theinclination angle θ is in the range of 10° to 80°. From a viewpoint ofimproving the resolution of an observed image, it is preferable that theinclination angle θ be in the range of 20° to 70°. In addition, from aviewpoint of improving the resolution of an observed image and thestability of a field of view, the inclination angle θ is more preferablyin the range of 30° to 65°.

The image acquiring unit 6, as illustrated in FIG. 1, is a device thatacquires a plurality of partial image data corresponding to a part of anoptical image according to the observation light L3 formed by theimaging optical system 5. The image acquiring unit 6, for example, isconfigured to include an imaging device that captures an optical imageaccording to the observation light L3. As the imaging device, forexample, there is an area image sensor such as a CMOS image sensor or aCCD image sensor. Such an area image sensor is disposed on an imageformation face according to the imaging optical system 5, and, forexample, captures an optical image using a global shutter or a rollingshutter, and outputs data of a two-dimensional image to the computer 7.

For a method for acquiring partial image data of an optical imageaccording to the observation light L3, various forms may be employed.For example, as illustrated in FIG. 3(A), a sub array may be set on theimaging surface of the area image sensor 21. In reading a sub array inthe area image sensor, only a set pixel column can be read among all thepixel columns, and a frame rate can be improved. Accordingly, in thiscase, only a pixel column 21 a included in the sub array can be read,and accordingly, partial image data can be acquired by capturing a partof the optical image according to the observation light L3. In addition,as illustrated in FIG. 3(B), partial image data may be acquired bysetting all the pixel columns of the area image sensor 21 as a read areaand extracting a part of a two-dimensional image using image processingperformed thereafter.

Furthermore, as illustrated in FIG. 3(C), partial image data may beacquired by limiting the imaging surface to one pixel column using aline sensor 22 instead of the area image sensor 21. In addition, asillustrated in FIG. 3(D), by disposing a slit 23 transmitting only apart of the observation light L3 on a front face of the area imagesensor (an optical detector) 21, image data of the pixel column 21 acorresponding to the slit 23 may be acquired as partial image data.Furthermore, in a case in which the slit 23 is used, instead of the areaimage sensor 21, a point sensor such as a photomultiplier tube may beused.

The computer 7 is physically configured to include a memory such as aRAM, a ROM, and the like, a processor (an arithmetic operation circuit)such as a CPU, a communication interface, a storage unit such as a harddisk, and, a display unit such as a display. As such a computer 7, forexample, there is a personal computer, a cloud server, a smart device (asmartphone, a tablet terminal, or the like), a microcomputer, or thelike. By executing a program stored in the memory using the CPU of thecomputer system, the computer 7 functions as a controller controllingoperations of the light source 2 and the moving stage 12, an imagegenerating unit 8 generating observation image data of a sample S, andan analysis unit 10 analyzing the observation image data (see FIG. 1).

The computer 7, as a controller, receives an input of a user'smeasurement start operation and drives the light source 2, the movingstage 12, and the image acquiring unit 6 in synchronization with eachother. In this case, during moving of the sample S according to themoving stage 12, the computer 7 may perform control of the light source2 such that the light source 2 continuously outputs the light L1 or maycontrol on/off of an output of the light L1 using the light source 2 inaccordance with imaging executed by the image acquiring unit 6. Inaddition, in a case in which the emission optical system 3 includes anoptical shutter (not illustrated in the drawing), the computer 7 mayturn on/off emission of the planar light L2 onto the sample S bycontrolling the optical shutter.

In addition, the computer 7, as the image generating unit 8, generatesobservation image data of the sample S based on a plurality of partialimage data generated by the image acquiring unit 6. The image generatingunit 8 generates observation image data of the sample S on a face (theXY plane) orthogonal to the optical axis P1 of the planar light L2, forexample, based on the plurality of partial image data output from theimage acquiring unit 6. The image generating unit 8 executes storage ofthe generated observation image data, display of the observation imagedata on a monitor or the like, and the like in accordance with a user'spredetermined operation.

The computer 7, as the analysis unit 10, generates an analysis result byexecuting an analysis based on the observation image data generated bythe image generating unit 8. The analysis unit 10 executes storage ofthe generated analysis result, display of the analysis result on a motoror the like, and the like in accordance with a user's predeterminedoperation. In addition, only the analysis result generated by theanalysis unit 10 may be displayed on a monitor or the like withoutperforming display of the observation image data generated by the imagegenerating unit on a monitor or the like.

[Sample Observation Method]

FIG. 4 is a flowchart illustrating one example of a sample observationmethod using a sample observation device. As illustrated in the drawing,this sample observation method includes an emission step (Step S01), ascanning step (Step S02), an image forming step (Step S03), an imageacquiring step (Step S04), an image generating step (S05), and ananalysis step (Step S06).

In the emission step S01, the planar light L2 is emitted to a sample S.When a measurement start operation is input by a user, the light sourceis driven based on a control signal supplied from the computer 7, andlight L1 is output from the light source 2. The light L1 output from thelight source 2 is shaped by the emission optical system 3 to be planarlight L2, and the planar light L2 is emitted to the sample S.

In the scanning step S02, the sample S is scanned by the emission face Rof the planar light L2. When a measurement start operation is input by auser, the moving stage 12 is driven in synchronization with driving ofthe light source 2 based on a control signal supplied from the computer7. Accordingly, the sample container 11 is linearly driven at a constantspeed in the Y-axis direction, and the sample S disposed inside the well13 is scanned by the emission face R of the planar light L2.

In the image forming step S03, by using the imaging optical system 5having an observation axis P2 inclined with respect to the emission faceR, an image of the observation light L3 generated in the sample Saccording to the emission of the planar light L2 is formed on the imageformation face of the image acquiring unit 6. In the image acquiringstep S04, a plurality of partial image data corresponding to a part ofan optical image according to the observation light L3 that is formed bythe imaging optical system 5 are acquired. The partial image data issequentially output from the image acquiring unit 6 to the imagegenerating unit 8.

In the image generating step S05, observation image data of the sample Sis generated based on the plurality of partial image data. In thisembodiment, as illustrated in FIGS. 1 and 2, the emission face R of theplanar light L2 for the sample S is a face within an XZ plane, and thesample S is scanned with the emission face R in the Y-axis direction.Accordingly, as illustrated in FIG. 5(A), in accordance with acquisitionof a plurality of pieces of XZ cross-sectional image data 31 that ispartial image data in the Y-axis direction, three-dimensionalinformation of the sample S is accumulated in the image generating unit8. In the image generating unit 8, data is rebuilt using a plurality ofXY cross-sectional images, and, for example, as illustrated in FIG.5(B), an XY cross-sectional image having an arbitrary thickness at anarbitrary position of the sample S in the Z axis direction is generatedas observation image data 32 in which a background is suppressed.

In the analysis step S06, the observation image data is analyzed by theanalysis unit 10, and an analysis result is generated. For example, indrug discovery screening, a sample S and a reagent are put into thesample container 11, and observation image data is acquired. Then, theanalysis unit 10 evaluates the reagent based on the observation imagedata and generates evaluation data as a result of the analysis.

Effects

A sample observation device 100 according to a comparative example, asillustrated in FIG. 6(A), has an observation axis P2 that is orthogonalto the emission face R of the planar light L2. In this sampleobservation device 100, by emitting the planar light L2 onto the wholesurface of a focused face of the observation optical system, an image ofa fault plane orthogonal to the direction of the observation axis P2 inthe sample S can be acquired by performing imaging once. Accordingly, inorder to acquire three-dimensional information of the sample S, it isnecessary to acquire images of a plurality of fault planes orthogonal tothe direction of the observation axis P2 by scanning the sample S in thedirection of the observation axis P2. The sample observation device 100according to the comparative example, as illustrated in FIG. 6(B), needsto repeat selection of a fault plane from which an image is acquired(scanning of the sample S and stopping) and image acquisition until theimages of all the fault planes are acquired. In addition, in a case inwhich an area in which an observation target is present is larger thanan imaging area, in addition to the operation of acquiring across-sectional image in the direction of the observation axis P2, anoperation of selecting an imaging field of view and the like inaccordance with movement of a stage in a direction different from theobservation axial direction are necessary.

In contrast to this, in the sample observation device 1 according to anexample, as illustrated in FIG. 7(A), while a sample S is scanned withthe emission face R of the planar light L2, image acquisition isperformed by the image acquiring unit 6, and the observation axis P2 ofthe imaging optical system 5 is inclined with respect to the emissionface R of the planar light L2. For this reason, the image acquiring unit6 can sequentially acquire partial image data of fault planes in thedirection of the optical axis P1 (the Z-axis direction) of the planarlight L2, and the image generating unit 8 can generate observation imagedata 32 of the sample S based on a plurality of partial image data.

In this sample observation device 1, as illustrated in FIG. 7(B), imageacquisition can be sequentially performed while the sample S is scanned.In the operation of the sample observation device 100 according to thecomparative example, every time when the moving stage 12 is driven andstopped, a time loss occurs in accordance with an influence of inertiaand the like. On the other hand, in the sample observation device 1, thenumber of times of driving and stopping of the moving stage 12 isdecreased, and a scanning operation for a sample and image acquisitionare simultaneously performed, whereby the throughput until theacquisition of the observation image data 32 is improved.

In addition, in the sample observation device 1, as illustrated in FIG.2, a sample S is held by the sample container 11 having the input face15 a of the planar light L2, and the optical axis P1 of the planar lightL2 according to the emission optical system 3 is arranged to beorthogonal to the input face 15 a of the sample container 11. Inaddition, in the sample observation device 1, the scanning unit 4 scansthe sample S in a direction (the Y-axis direction) orthogonal to theoptical axis P1 (Z-axis direction) of the planar light L2 according tothe emission optical system 3. Accordingly, image processing such as aposition correction for the partial image data acquired by the imageacquiring unit 6 and the like is not necessary, and the process ofgenerating observation image data can be easily performed.

In addition, in the sample observation device 1, the inclination angle θof the observation axis P2 of the imaging optical system 5 for theemission face R of the planar light L2 in the sample is in the range of10° to 80°, is preferably in the range of 20° to 70°, and is morepreferably in the range of 30° to 65°. Hereinafter, this point will bereviewed.

FIG. 8 is a diagram illustrating an example of calculation of a field ofview in a sample observation device. In the example illustrated in thedrawing, an imaging optical system is positioned in a medium A having arefractive index n1, and an emission face of planar light is positionedin a medium B having a refractive index n2. In a case in which a fieldof view in the imaging optical system is denoted by V, an emission faceis denoted by V′, an inclination angle of the observation axis for theemission face is denoted by θ, a refraction angle at an interfacebetween the media A and B is denoted by θ′, and a distance on theinterface between the media A and B at the inclination angle θ of thefield of view V is denoted by L, the following Equations (1) to (3) aresatisfied.

(Math 1)

L=V/cos θ  (1)

(Math 2)

sin θ′=(n1/n2)sin θ  (2)

(Math 3)

V′=L/tan θ′  (3)

FIG. 9 is a diagram illustrating a relation between an inclination angleof an observation axis and resolution. In the drawing, the horizontalaxis represents the inclination angle θ of the observation axis, and thevertical axis represents a relative value V′/V of the field of view.Then, a value of V′/V acquired when the refractive index n1 of themedium A is set to “1” (air), and the refractive index n2 of the mediumB is changed from 1.0 to 2.0 at the interval of 0.1 is plotted withrespect to the inclination angle θ. It is represented that theresolution in the depth direction (hereinafter, referred to as“Z-direction resolution”) of the sample is higher as the value of V′/Vbecomes smaller, and the Z-direction resolution is lower as the valuebecomes larger.

From the result illustrated in FIG. 9, in a case in which the refractiveindex n1 of the medium A and the refractive index n2 of the medium arethe same, it can be understood that the value of V′/V is inverselyproportional to the inclination angle θ. In addition, in a case in whichthe refractive index n1 of the medium A and the refractive index n2 ofthe medium B are different from each other, it can be understood thevalue of V′/V forms a parabola with respect to the inclination angle θ.From this result, it can be understood that the Z-direction resolutioncan be controlled using the refractive index of a space in which thesample is arranged, a refractive index of a space in which the imagingoptical system is arranged, and the inclination angle θ of theobservation axis. In addition, it can be understood that higher.Z-direction resolution is acquired in the range of 10° to 80° of theinclination angle θ than in a range in which the inclination angle θ isless than 10° or more than 80°.

In addition, from the result illustrated in FIG. 9, it can be understoodthat the inclination angle θ at which the Z-direction resolution is amaximum tends to be lower as a difference between the refractive indexn1 and the refractive index n2 becomes larger. In a case in which therefractive index n2 is in the range of 1.1 to 2.0, the inclination angleθ at which the Z-direction resolution is a maximum is in the range ofabout 47° to about 57°. For example, in a case in which the refractiveindex n2 is 1.33 (water), the inclination angle θ at which theZ-direction resolution is a maximum is estimated to be about 52°. Inaddition, for example, in a case in which the refractive index n2 is1.53 (glass), the inclination angle θ at which the Z-directionresolution is a maximum is estimated to be about 48°.

FIG. 10 is a diagram illustrating a relation between the inclinationangle of the observation axis and stability of the field of view. In thedrawing, the horizontal axis represents the inclination angle θ of theobservation axis, and the vertical axis represents the stability of thefield of view. The stability is represented as a ratio between adifference between V′/V at an inclination angle θ+1 and V′/V at aninclination angle θ and a difference between V′/V at an inclinationangle θ−1 and V′/V at the inclination angle θ and is calculated based onthe following Equation (4). As the stability is closer to 0%, it can beevaluated that a change in the field of view with respect to a change inthe inclination angle is small, and the view of field is stabilized. Inthis FIG. 10, similar to FIG. 9, the stability acquired when therefractive index n1 of the medium A is set to “1” (air), and therefractive index n2 of the medium B is changed from 1.0 to 2.0 at theinterval of 0.1 is plotted.

(Math 4)

Stability (%)=((V′/V)_(θ+1)−(V′/V)_(θ−1))/(V′/V)₀  (4)

From the result illustrated in FIG. 10, it can be understood that, in arange in which the inclination angle θ is less than 10° or more than80°, the stability exceeds +20%, and it is difficult to control the viewof field. On the other hand, in a case in which the angle θ is in therange of 10° to 80°, the stability is equal to or less than ±20%, andthe field of view can be controlled. In addition, in a case in which theinclination angle θ is in the range of 20° to 70°, the stability isequal to or less than ±10%, and the field of view can be easilycontrolled.

FIG. 11 is a diagram illustrating a relation between the inclinationangle of the observation axis and transmittance of observation lighttransmitted from a sample. In the drawing, the horizontal axisrepresents the inclination angle θ of the observation axis, a leftvertical axis represents a relative value of the field of view, and aright vertical axis represents transmittance. In this FIG. 11, inconsideration of the maintaining state of a sample in the samplecontainer, the refractive index n1 of the medium A is set to 1 (air),the refractive index n2 of the medium B is set to 1.53 (glass), and therefractive index n3 of the medium C is set to 1.33 (water), and a valueof transmittance is product of transmittance of an interface between themedia B and C and transmittance of an interface between the media A andB. In FIG. 11, the transmittance of P waves, the transmittance of Swaves, and the dependency of an average value thereof on the angle areplotted. In addition, in FIG. 11, a relative value of the field of viewin the medium C is plotted together.

From the result illustrated in FIG. 11, it can be understood that thetransmittance of observation light from a sample to the imaging opticalsystem is changeable by changing the inclination angle θ of theobservation axis. It can be understood that at least 50% or highertransmittance is acquired in a range in which the inclination angle θ isequal to or less than 80°. In addition, it can be understood that atleast 60% or higher transmittance is acquired in a range in which theinclination angle θ is equal to or less than 70°, and at least 75% orhigher transmittance is acquired in a range in which the inclinationangle θ is equal to or less than 65°.

From the results described above, in a case in which the Z-directionresolution of a sample is requested, for example, it is appropriate toselect the inclination angle θ from the range of 30° to 65° such thatthe value of V′/V, which is a relative value of the field of view, isequal to or less than 3, the stability is less than 5%, and thetransmittance (an average value of P waves and S waves) of theobservation light is equal to or higher than 75%. On the other hand, ina case in which the Z-direction resolution of a sample is not requested,the inclination angle θ may be appropriately selected from a range of10° to 80°, and, from a viewpoint of securing a range of the field ofview per pixel, it is appropriate to select the inclination angle θ froma range of 10° to 30° or 65° to 80°.

The sample observation device and the sample observation method are notlimited to those according to the embodiment described above. Forexample, the optical axis P1 of the planar light L2 and the input face15 a of the sample container 11 may not necessarily be orthogonal toeach other, and the optical axis P1 of the planar light L2 and thescanning direction of the sample S scanned by the scanning unit 4 maynot necessarily be orthogonal to each other.

In addition, for example, in the embodiment described above, althoughthe transparent member 15 is disposed to occupy the one end side of thewell 13 in the sample container 11, and the planar light L2 is inputfrom the input face 15 a of the transparent member 15, the planar lightL2 may be configured to be input from the other end side of the well 13.In such a case, the number of interfaces between media having differentrefractive indexes is decreased, and the number of times of refractionof the observation light L3 can be decreased. In addition, instead ofthe sample container 11, a sample S may be maintained in a solid such asgel, and, like a flow cytometer, a sample S may move by causing a fluidbody such as a sheath liquid to flow inside the transparent container.In the case of the flow cytometer, a sheath liquid in which a liquidcontaining a test body that is a sample S is included flows inaccordance with a flow cell. Accordingly, the test body moves whilebeing arranged, and accordingly, the flow cell can be positioned as ascanning unit.

In addition, a plurality of imaging optical system 5 and a plurality ofimage acquiring units 6 may be disposed. In such a case, in addition toenlargement of the observation range, a plurality of pieces ofobservation light L3 having different wavelengths can be observed. Inaddition, a plurality of image acquiring units 6 may be disposed for oneimaging optical system 5, and one image acquiring unit 6 may be disposedfor a plurality of imaging optical systems 5. A plurality of imageacquiring units 6 may combine optical detectors or imaging devices ofdifferent types. The light source 2 may be configured by a plurality oflight sources outputting light having different wavelengths. In such acase, excitation light having different wavelengths can be emitted to asample S.

In addition, in order to alleviate astigmatism, a prism may be disposedin the imaging optical system 5. In such a case, for example, asillustrated in FIG. 12, the prism 41 may be disposed on a later stageside of the objective lens 16 (between the objective lens 16 and theimage acquiring unit 6). For a countermeasure for defocusing, theimaging surface of the imaging device in the image acquiring unit 6 maybe inclined with respect to the observation axis P2. Furthermore, aconfiguration may be employed in which separation of wavelengths of theobservation light L3 may be performed by disposing a dichroic mirror ora prism, for example, between the imaging optical system 5 and the imageacquiring unit 6.

In addition, as described above, as the observation light L3,fluorescent light excited by the planar light L2, scattered light of theplanar light L2, diffuse reflected light of the planar light L2, or thelike may be considered. For this reason, the image acquiring unit 6 mayacquire image data of the observation light L3 of different types. Insuch a case, at least two types of observation light among fluorescentlight excited by the planar light L2, scattered light of the planarlight L2, and diffuse reflected light of the planar light L2 may be setas targets.

REFERENCE SIGNS LIST

-   -   1 Sample observation device    -   3 Emission optical system    -   4 Scanning unit    -   5 Imaging optical system    -   6 Image acquiring unit    -   8 Image generating unit    -   10 Analysis unit    -   11 Sample container    -   15 a Input face    -   21 Area image sensor (imaging device)    -   22 Line sensor    -   23 Slit    -   31 Partial image data    -   32 Observation image data    -   L2 Planar light    -   L3 Observation light    -   P2 Observation axis    -   R Emission face    -   S Sample    -   θ Inclination angle

1. A system comprising: an emission optical system configured to emitplanar light onto a sample; a scanner configured to scan the sample withrespect to an emission face of the planar light; an imaging opticalsystem having an observation axis inclined with respect to the emissionface and configured to form an image from observation light generated inthe sample in accordance with the emission of the planar light; an imageacquiring unit configured to acquire a plurality of partial image datacorresponding to a part of an optical image according to the observationlight formed as an image by the imaging optical system; and a computerconfigured to generate observation image data of the sample based on theplurality of partial image data generated by the image acquiring unit.2. The system according to claim 1, further comprising: a samplecontainer having an input face of the planar light and configured tohold the sample, and wherein an optical axis of the planar lightaccording to the emission optical system is disposed to be orthogonal tothe input face of the sample container.
 3. The system according to claim1, wherein the scanner is configured to scan the sample in a directionorthogonal to the optical axis of the planar light according to theemission optical system.
 4. The system according to claim 1, wherein aninclination angle of the observation axis of the imaging optical systemwith respect to the emission face of the planar light is in the range of10° to 80°.
 5. The system according to claim 1, wherein an inclinationangle of the observation axis of the imaging optical system with respectto the emission face of the planar light is in the range of 20° to 70°.6. The system according to claim 1, wherein an inclination angle of theobservation axis of the imaging optical system with respect to theemission face of the planar light is in the range of 30° to 65°.
 7. Thesystem according to claim 1, wherein the image acquiring unit includes atwo-dimensional image sensor and is configured to extract image datacorresponding to a part of the optical image of the observation lightfrom data output from the two-dimensional image sensor as the partialimage data.
 8. The system according to claim 1, wherein the imageacquiring unit includes a line sensor for capturing configured tocapture a part of the optical image according to the observation lightand outputting the partial image data.
 9. The system according to claim1, wherein the image acquiring unit includes a slit transmitting a partof an optical image according to the observation light and an opticaldetector for detecting configured to detect an optical image transmittedthrough the slit and is configured to generate the partial image databased on data output from the optical detector.
 10. The system accordingto claim 1, wherein the image generating unit computer is configured togenerate observation image data of the sample on a face orthogonal tothe optical axis of the planar light based on the plurality of partialimage data.
 11. The system according to a claim 1, further comprising ananalysis unit for analyzing analyzer configured to analyze theobservation image data and generating to generate an analysis result.12. A method comprising: emitting planar light onto a sample; scanningthe sample with respect to an emission face of the planar light; formingan image from observation light generated in the sample in accordancewith the emission of the planar light using an imaging optical systemhaving an observation axis inclined with respect to the emission face;acquiring a plurality of partial image data corresponding to a part ofan optical image according to the observation light formed as an imageby the imaging optical system; and generating observation image data ofthe sample based on the plurality of partial image data.
 13. The methodaccording to claim 12, wherein an optical axis of the planar light isdisposed to be orthogonal to an input face of a sample containerconfigured to hold the sample.
 14. The method according to claim 12,wherein the scanning scans the sample in a direction orthogonal to theoptical axis of the planar light.
 15. The method according to claim 12,wherein an inclination angle of the observation axis of the imagingoptical system with respect to the emission face of the planar light isin the range of 10° to 80°.
 16. The method e according to claim 12,wherein an inclination angle of the observation axis with respect to theemission face of the planar light is in the range of 20° to 70°.
 17. Themethod according to claim 12, wherein an inclination angle of theobservation axis with respect to the emission face of the planar lightis in the range of 30° to 65°.
 18. The method according to claim 12,wherein the acquiring extracts image data corresponding to a part of theoptical image of the observation light from data output from atwo-dimensional image sensor as the partial image data.
 19. The methodaccording to claim 12, wherein acquiring captures a part of the opticalimage according to the observation light by a line sensor to output thepartial image data.
 20. The method according to claim 12, wherein theacquiring transmits a part of an optical image according to theobservation light by a slit and detecting an optical image transmittedthrough the slit to generate the partial image data.
 21. The methodaccording to claim 12, wherein the generating generates observationimage data of the sample on a face orthogonal to the optical axis of theplanar light based on the plurality of partial image data.
 22. Themethod according to claim 12, further comprising: analyzing theobservation image data to generate an analysis result.